Electrophoresis buffer for extending the useful electrophoresis life of an electrophoresis gel

ABSTRACT

There is provided an electrolyte solution for extending useful electrophoresis life of an electrophoresis gel containing Tris(hydroxymethyl)aminomethane (TRIS), at least one zwitterion, and water. The electrolyte solution may be used in buffer systems for gel electrophoresis, such as SDS-PAGE.

This application claims priority under 35USC §120 from and the benefitof U.S. patent application Ser. No. 14/571,865, which is a Continuationof U.S. patent application Ser. No. 13/547,633 filed on Jul. 12, 2012,which claims priority from and the benefit of U.S. ProvisionalApplication No. 61/507,883, filed Jul. 14, 2011, the specifications ofwhich are hereby incorporated by reference in their entireties.

BACKGROUND

(a) Field

The subject matter disclosed generally relates to gel electrophoresis.More specifically, the subject matter disclosed relates to anelectrolyte solution for extending the useful electrophoresis life of anelectrophoresis gel during gel electrophoresis containing at least onezwitterion, and water.

(b) Related Prior Art

Gel electrophoresis is a common procedure for the separation ofbiological molecules, such as deoxyribonucleic acid (DNA), ribonucleicacid (RNA), polypeptides and proteins. In gel electrophoresis, themolecules are separated into bands according to the rate at which animposed electric field causes them to migrate through a filtering gel.

The basic apparatus used in this technique consists of a gel enclosed ina glass tube, sandwiched as a slab between glass or plastic plates, orpoured in a plastic tray. The gel has an open molecular networkstructure, defining pores which are saturated with an electricallyconductive buffered solution. These pores through the gel are largeenough to admit passage of the migrating macromolecules.

The gel is placed in a chamber in contact with buffer solutions whichmake electrical contact between the gel and the cathode or anode of anelectrical power supply. A sample containing the macromolecules and atracking dye is placed on top of the gel. An electric potential isapplied to the gel causing the sample macromolecules and tracking dye tomigrate toward the bottom of the gel. The electrophoresis is halted justbefore the tracking dye reaches the end of the gel.

The most common buffer system employed for the separation of proteins isthe Laemmli buffer system consists of 0.375 M tris (hydroxy methyl)amino-methane (Tris), titrated to pH 8.8 with HCl, in the separatinggel. The stacking gel consists of 0.125 M Tris, titrated to pH 6.8. Theanode and cathode running buffers contain 0.024 M Tris, 0.192 M glycine,0.1% SDS. Many different gel separation materials have been disclosed,with different compositions, pH characteristics, voltage requirements,etc. The goal of most of the recent innovations in the field has been toprovide an electrophoresis gel which can be used to perform a faster,more accurate, more stable, or therefore more versatile electrophoresis.

A number of different gel buffer systems have been proposed for use ator around neutral pH that do not involve the use of the Tris-HCl Glycinebuffer system of Laemmli.

For example, U.S. Pat. No. 6,096,182 to Updyke et al. discloses anelectrophoresis gel at a neutral pH. The advantage of producing such agel is that the gel system is stable, with reduced reactivity andincreased shelf life.

U.S. Pat. No. 5,464,517 to Hjerten et al. discloses an electrophoresisbuffer which has a high buffering capacity and low electricalconductivity. The advantage of this type of buffer, particularly incapillary electrophoresis, is that it allows the separation to beperformed at a higher voltage and consequently more quickly.

A majority of innovations have focused on improving electrophoresis byproposing new recipes for the gel buffer.

Currently, a major obstacle in the production and sale of pre-castelectrophoresis gels is their rather short shelf life of about 3 months.For example, for pre-cast polyacrylamide gels, it is believed that theirdegradation is a consequence of the hydrolysis of amide groups to formpartially anionic carboxylic acid derivatives under basic conditions.Therefore, the high pH (e.g. pH 8.0 to 9.5) is believed to lower thestability of the gels upon storage. The hydrolysis is believed to leadto loss of resolution of the separated molecules, reduced migrationdistance of the separated molecule, and the reduced intensity of proteinstaining.

Therefore, there is a need for reagents that will extend the usefulelectrophoresis life of gels that have gone pass their normal expirydate (shelf-life).

SUMMARY

In a first embodiment there is disclosed an electrolyte solution forextending a useful electrophoresis life of an electrophoresis gelcomprising:

-   -   at least one zwitterion chosen from        2-amino-2methyl-1,3-propanediol (AMPD),        N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic        acid (AMPSO), N-Glycylglycine (Gly-Gly),        4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or        HEPPS), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),        3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO),        2-(cyclohexylamino)ethanesulfonic acid (CHES),        N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),        (2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesulphonic        acid) (TES), N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic        acid (TAPS) and 3-N-Morpholino propanesulfonic acid (POPSO); and    -   water.

The zwitterion may be 4-(2-hydroxyethyl)piperazine-1-propanesulfonicacid (EPPS or HEPPS).

The zwitterion may be chosen from N-Glycylglycine (Gly-Gly) and3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO).

The pH is of the electrolyte solution may be from about 8.0 to about 11.

The electrolyte solution may be further comprisingTris(hydroxymethyl)aminomethane (TRIS).

The electrolyte solution may be further comprising sodium dodecylsulphate (SDS).

The electrolyte solution may further be comprising a chelating agenthaving the name: ethylenediaminetetraacetate (EDTA), ethylene glycoltetraacetic acid (EGTA), trisodium nitrilotriacetate, hydroxyethylethylenediamine trisodium acetate (trisodium HEDTA), diethylene triaminopentasodium acetate or uramil disodium acetate.

The concentration of Tris(hydroxymethyl)aminomethane (TRIS) may be fromabout 10 mM to about 500 mM.

The concentration of Tris(hydroxymethyl)aminomethane (TRIS) may be fromabout 50 mM to about 150 mM.

The concentration of Tris(hydroxymethyl)aminomethane (TRIS) may be fromabout 50 mM to about 300 mM.

The concentration of Tris(hydroxymethyl)aminomethane (TRIS) may be 150mM.

The concentration of the zwitterion may be from about 1 mM to about 500mM.

The concentration of the zwitterion may be from about 10 mM to about 500mM.

The concentration of the zwitterion may be from about 25 mM to about 75mM.

The concentration of the zwitterion may be from about 50 mM to about 100mM.

The concentration of the zwitterion may be 50 mM.

The concentration of the zwitterion may be 100 mM.

The concentration of sodium dodecyl sulphate (SDS) may be 0.5% (wt/vol)or less.

The concentration of sodium dodecyl sulphate (SDS) may be 0.1% (wt/vol)or less.

The concentration of sodium dodecyl sulphate (SDS) may be 0.1% (wt/vol).

The concentration of ethylenediaminetetraacetate (EDTA) may be 0.5%(wt/vol) or less.

The concentration of ethylenediaminetetraacetate (EDTA) may be 0.05%(wt/vol) or less.

The concentration of ethylenediaminetetraacetate (EDTA) may be 0.03%(wt/vol).

According to another embodiment, there is disclosed a method ofextending a useful electrophoresis life of an electrophoresis gel duringan electrophoretic separation of at least one sample comprising the stepof:

-   -   applying a voltage to an electrolyte solution according to the        present invention in contact with an electrophoresis gel        containing the at least one sample therein.

According to another embodiment, there is disclosed a method ofextending a useful electrophoresis life of an electrophoresis gel duringan electrophoretic separation of at least one sample comprising the stepof:

-   -   adding at least one zwitterion chosen from        2-amino-2methyl-1,3-propanediol (AMPD),        N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic        acid (AMPSO), N-Glycylglycine (Gly-Gly),        4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or        HEPPS), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),        3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO),        2-(cyclohexylamino)ethanesulfonic acid (CHES),        N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),        (2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesulphonic        acid) (TES), N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic        acid (TAPS) and 3-N-Morpholino propanesulfonic acid (POPSO) to        an electrophoresis buffer.

The zwitterion may be 4-(2-hydroxyethyl)piperazine-1-propanesulfonicacid (EPPS or HEPPS).

The zwitterion may be chosen from N-Glycylglycine (Gly-Gly) and3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO).

The method of the present invention may be further comprising the stepof applying a voltage to the electrophoresis buffer in contact with anelectrophoresis gel containing the at least one sample therein.

The following terms are defined below.

The term “improved resolution” is intended to mean a better resolutionwhich allows separation of sharper or narrower bands of molecules,distanced or spaced apart from each other as opposed to other means ofseparation which have broader or thicker bands. This facilitatesphysical separation or molecular weight identification of the differentmolecules that make up these bands over the entire range of molecularweight.

The term “useful electrophoresis life” is intended to mean the normaloperating life of an electrophoresis gel in terms of utility to theowner. This covers the period of time during which there is nounacceptable loss of electrophoretic quality of the electrophoresis gel(e.g. loss of resolution, decrease in migration speed, migrationartefacts, over-heating polyacrylamide gel, gel cracking or distortion,etc.); and during which the electrophoresis gel remains usable for itsintended purpose.

Features and advantages of the subject matter hereof will become moreapparent in light of the following detailed description of selectedembodiments, as illustrated in the accompanying figures. As will berealized, the subject matter disclosed and claimed is capable ofmodifications in various respects, all without departing from the scopeof the claims. Accordingly, the drawings and the description are to beregarded as illustrative in nature, and not as restrictive and the fullscope of the subject matter is set forth in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates polyacrylamide gels electrophoresed in electrolytesolutions according embodiments of the present invention and compared toa gel electrophoresed on Tris-glycine-SDS (baseline) solution. All thegels electrophoreses were pass their expiry date.

FIG. 2 illustrates polyacrylamide gels electrophoresed in electrolytesolutions according embodiments of the present invention and compared toa gel electrophoresed on Tris-glycine-SDS (baseline) solution. All thegels electrophoreses were pass their expiry date.

FIG. 3 illustrates polyacrylamide gels electrophoresed in electrolytesolutions according embodiments of the present invention (A) andcompared to a gel electrophoresed on Tris-glycine-SDS (baseline)solution (B). All the gels electrophoresed were passed their expirydate.

FIG. 4 illustrates a polyacrylamide gel electrophoresed in electrolytesolutions according embodiments of the present invention. The gelelectrophoresed is passed its expiry date.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present inventors have now surprisingly found that selectingspecific zwitterions to prepare an electrolyte solution for use in gelelectrophoresis can result in the unexpected extension of the usefullife of electrophoresis gel for their use in electrophoresis (or as alsoused herein, electrophoretic separation). This new and unexpectedproperty is in addition to any other property that the specificzwitterions identified may have, for example provide increases inelectrophoresis speed, improvements in gel resolution, or both. Thisimprovement may be observed, for example, when the electrolyte solutionsare used as running buffer (also referred to as “reservoir” buffer) forthe electrophoresis apparatus in which electrophoresis gels that havegone pass their expected expiry date are employed for electrophoresis.

In embodiments there are disclosed electrolyte solutions for performinggel electrophoresis. The electrolyte contains specific components.

Zwitterions

A zwitterion is a chemical compound that carries a total net charge of 0and is thus electrically neutral, but carries formal charges ondifferent atoms. Zwitterions are polar and are usually verywater-soluble, but poorly soluble in most organic solvents. Zwitterionswill exist mostly as zwitterions in a certain range of pH. The pH atwhich the average charge is zero is known as the molecule's isoelectricpoint.

The zwitterions of interest in the present invention belong to thecategory commonly referred to as biological buffers, which are buffersthat are commonly used as buffering agents in biological laboratories.Examples of biological buffers that can be cited are those known asbis-TRIS (2-bis[2-hydroxyethyl]amino-2-hydroxymethyl-1,3-propanediol),ADA (N-[2-acetamido]-2-iminodiacetic acid), ACES(2-[2-acetamino[-2-aminoethanesulphonic acid), PIPES(1,4-piperazinediethanesulphonic acid), MOPSO(3-[N-morpholino]-2-hydroxypropanesulphonic acid), bis-TRIS PROPANE(1,3-bis[tris(hydroxymethyl)methylaminopropane]), BES(N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid), MOPS(3-[N-morpholino]propancsulphonic acid), TES(2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesulphonic acid),HEPES (N-[2-hydroxyethyl]piperazine-N′-(2-ethanesulphonic)acid), DIPSO(3-N,N-bis[2-hydroxyethyl]amino-2-hydroxypropanesulphonic acid), MOBS(4-N-morpholinobutanesulphouic acid), TAPSO(3[N-tris-hydroxymethyl-methylamino]-2-hydroxypropanesulphonic acid),TRIS (2-amino-2-[hydroxymethyl]-1,3-propanediol), HEPPSO(N-[2-hydroxyethyl]piperazine-N′-[2-hydroxypropanesulphonic]acid), POPSO(piperazie-N,N′-bis[2-hydroxypropanesulphonic]acid), TEA(triethanolamine), EPPS (or HEPPS)(N-[2-hydroxyethyl]-piperazine-N′-[3-propanesulphonic]acid), TRICINE(N-tris[hydroxymethyl]methylglycine), GLY-GLY (diglycine), BICINE(N,N-bis[2-hydroxyethyl]glycine), HEPBS(N-[2-hydroxyethyl]piperazine-N′-[4-butanesulphonic]acid), TAPS(N-tris[hydroxymethyl]methyl-3-aminopropanesulphonic acid), AMPD(2-amino-2-methyl-1,3-propanediol), TABS(N-tris[hydroxymethyl]methyl-4-aminobutanesulphonic acid), AMPSO(3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulphonic acid),CHES (2-(N-cyclohexylamino)ethanesulphonic acid), CAPSO(3-[cyclohexylamino]-2-hydroxy-1-propanesulphonic acid), AMP(2-amino-2-methyl-1-propanol), CAPS(3-cyclohexylamino-1-propanesulphonic acid), and CABS(4-[cyclohexylamino]-1-butanesulphonic acid).

Although the biological buffering property of these zwitterions has beenrecognized, the capacity of a select group of these zwitterions topositively impact the performance of gel electrophoresis, and the usefulelectrophoresis life of gels for gel electrophoresis was not.

Preferably the zwitterions include 2-amino-2methyl-1,3-propanediol(AMPD), N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonicacid (AMPSO), N-Glycylglycine (Gly-Gly),4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or HEPPS),3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO),2-(cyclohexylamino)ethanesulfonic acid (CHES),N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),(2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino]ethanesulphonic acid)(TES), N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS)and 3-N-Morpholino propanesulfonic acid (POPSO). These zwitterionssignificantly improve the useful electrophoresis life of electrophoresisgels that have gone pass their expiry date when the electrolytesolutions are used as running buffer (also referred to as “reservoir”buffer) for the electrophoresis apparatus in which electrophoresis isbeing performed. The zwitterion may also be added to existing runningbuffers. When using these zwitterions in the electrolyte solutionaccording to the present invention, it may be desirable to adjust the pHof the electrolyte solution (e.g using NaOH or HCl) to an optimal pHvalue for performing the electrophoresis.

According to another embodiment, these zwitterions may also improve thespeed at which electrophoresis may be performed (as compared to theclassical Tris-Glycine-SDS at pH 8.3 of Laemmli), improve resolution (ascompared to the classical Tris-Glycine-SDS at pH 8.3 of Laemmli) byimproving the definition (or sharpness), by providing thinner or narrowbands) of the molecules separated, by improving the separation betweenthe molecules (i.e. the distance between the individual bands, or byboth improving the definition (or sharpness) and separation of themolecules. Also, when used for the preparation of gels, increase in theshelf life of gels prepared using these electrolyte solutions. Thezwitterions may be used alone or in combination.

The ranges of concentration over which these zwitterions may be used forthe preparation of electrolyte solutions according to the presentinvention, for extending the useful electrophoresis life as discussedherein are from about 1 mM to about 500 mM, or from about 10 mM to about500 mM, or about 25 mM to about 500 mM, or about 50 mM to about 500 mM,or about 75 mM to about 500 mM, or about 100 mM to about 500 mM, orabout 150 mM to about 500 mM, or about 200 mM to about 500 mM, or about250 mM to about 500 mM, or about 300 mM to about 500 mM, or about 350 mMto about 500 mM, or about 400 mM to about 500 mM, or about 450 mM toabout 500 mM, 1 mM to about 450 mM, or from about 10 mM to about 450 mM,or about 25 mM to about 450 mM, or about 50 mM to about 450 mM, or about75 mM to about 450 mM, or about 100 mM to about 450 mM, or about 150 mMto about 450 mM, or about 200 mM to about 450 mM, or about 250 mM toabout 450 mM, or about 300 mM to about 450 mM, or about 350 mM to about450 mM, or about 400 mM to about 450 mM, 1 mM to about 400 mM, or fromabout 10 mM to about 400 mM, or about 25 mM to about 400 mM, or about 50mM to about 400 mM, or about 75 mM to about 400 mM, or about 100 mM toabout 400 mM, or about 150 mM to about 400 mM, or about 200 mM to about400 mM, or about 250 mM to about 400 mM, or about 300 mM to about 400mM, or about 350 mM to about 400 mM, 1 mM to about 375 mM, or from about10 mM to about 375 mM, or about 25 mM to about 375 mM, or about 50 mM toabout 375 mM, or about 75 mM to about 375 mM, or about 100 mM to about375 mM, or about 150 mM to about 375 mM, or about 200 mM to about 375mM, or about 250 mM to about 375 mM, or about 300 mM to about 375 mM, orabout 350 mM to about 375 mM, or 1 mM to about 350 mM, or from about 10mM to about 350 mM, or about 25 mM to about 350 mM, or about 50 mM toabout 350 mM, or about 75 mM to about 350 mM, or about 100 mM to about350 mM, or about 150 mM to about 350 mM, or about 200 mM to about 350mM, or about 250 mM to about 350 mM, or about 300 mM to about 350 mM, 1mM to about 300 mM, or from about 10 mM to about 300 mM, or about 25 mMto about 300 mM, or about 50 mM to about 300 mM, or about 75 mM to about300 mM, or about 100 mM to about 300 mM, or about 150 mM to about 300mM, or about 200 mM to about 300 mM, or about 250 mM to about 300 mM, 1mM to about 250 mM, or from about 10 mM to about 250 mM, or about 25 mMto about 250 mM, or about 50 mM to about 250 mM, or about 75 mM to about250 mM, or about 100 mM to about 250 mM, or about 150 mM to about 250mM, or about 200 mM to about 250 mM, 1 mM to about 200 mM, or from about10 mM to about 200 mM, or about 25 mM to about 200 mM, or about 50 mM toabout 200 mM, or about 75 mM to about 200 mM, or about 100 mM to about200 mM, or about 150 mM to about 200 mM, 1 mM to about 150 mM, or fromabout 10 mM to about 150 mM, or about 25 mM to about 150 mM, or about 50mM to about 150 mM, or about 75 mM to about 150 mM, or about 100 mM toabout 150 mM, 1 mM to about 100 mM, or from about 10 mM to about 100 mM,or about 25 mM to about 100 mM, or about 50 mM to about 100 mM, or about75 mM to about 100 mM, 1 mM to about 75 mM, or from about 10 mM to about75 mM, or about 25 mM to about 75 mM, or about 50 mM to about 75 mM, 1mM to about 50 mM, or from about 10 mM to about 50 mM, or about 25 mM toabout 50 mM, 1 mM to about 25 mM, or from about 10 mM to about 25 mM, orfrom about 1 mM to about 10 mM, or from about 25 mM to about 50 mM, orfrom about 25 mM to about 100 mM, or from about 10 mM to about 100 mM,or from about 1 mM to about 100 mM, or from about 1 mM to about 75 mM,or from about 10 mM to about 75 mM, or from about 1 mM to about 10 mM,or from about 1 mM to about 50 mM or from about 10 mM to about 50 mM, orfrom about 25 mM to about 375 mM, and preferably, at 100 mM.

Optimal results with 4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid(EPPS or HEPPS) are obtained at pH of about pH 8.0 to 10.5.

Tris(hydroxymethyl)aminomethane base (TRIS)

Tris is an abbreviation of the organic compound known astris(hydroxymethyl)aminomethane, with the formula (HOCH₂)₃CNH₂. Tris isextensively used in biochemistry and molecular biology. In biochemistry,Tris is widely used as a component of buffer solutions, such as in TAEand TBE buffer, especially for solutions of nucleic acids and is thebasic element of the Laemmli buffer widely used in proteinelectrophoresis in Tris-Glycine buffers.

Tris also allows the pH of the electrolyte solution of the presentinvention to be set towards more basic pH values. According to oneembodiment, an electrolyte solution according to the present inventioncontaining EPPS at 47.5 mM may have a pH of about 5.9. The addition ofTris (about 150 mM) increases the pH at about 8.7. Tris also has apositive impact on the migration speed in the preferred concentrationranges.

Tris may be absent from the electrolyte solution of the presentinvention. However, the ranges of concentration over which Tris base maybe used for the preparation of electrolyte solutions according to thepresent invention are from about 0 mM to about 500 mM. The Trisconcentration has a direct impact on the pH of the solution. Anincreasing amount of Tris increases the pH. This allow fine adjustmentsin adapting the electrolyte solution of the present invention todifferent gel chemistry and keeping a proper pH ratio compatible withgel and buffer. The concentrations of Tris may be from about 0 mM toabout 500 mM, or from about 10 mM to about 500 mM, or about 25 mM toabout 500 mM, or about 50 mM to about 500 mM, or about 75 mM to about500 mM, or about 100 mM to about 500 mM, or about 150 mM to about 500mM, or about 200 mM to about 500 mM, or about 250 mM to about 500 mM, orabout 300 mM to about 500 mM, or about 350 mM to about 500 mM, or about400 mM to about 500 mM, or about 450 mM to about 500 mM, or from about 0mM to about 450 mM, or from about 10 mM to about 450 mM, or about 25 mMto about 450 mM, or about 50 mM to about 450 mM, or about 75 mM to about450 mM, or about 100 mM to about 450 mM, or about 150 mM to about 450mM, or about 200 mM to about 450 mM, or about 250 mM to about 450 mM, orabout 300 mM to about 450 mM, or about 350 mM to about 450 mM, or about400 mM to about 450 mM, or from about 0 mM to about 400 mM, or fromabout 10 mM to about 400 mM, or about 25 mM to about 400 mM, or about 50mM to about 400 mM, or about 75 mM to about 400 mM, or about 100 mM toabout 400 mM, or about 150 mM to about 400 mM, or about 200 mM to about400 mM, or about 250 mM to about 400 mM, or about 300 mM to about 400mM, or about 350 mM to about 400 mM, or from about 0 mM to about 375 mM,or from about 10 mM to about 375 mM, or about 25 mM to about 375 mM, orabout 50 mM to about 375 mM, or about 75 mM to about 375 mM, or about100 mM to about 375 mM, or about 150 mM to about 375 mM, or about 200 mMto about 375 mM, or about 250 mM to about 375 mM, or about 300 mM toabout 375 mM, or about 350 mM to about 375 mM, or from about 0 mM toabout 350 mM, or from about 10 mM to about 350 mM, or about 25 mM toabout 350 mM, or about 50 mM to about 350 mM, or about 75 mM to about350 mM, or about 100 mM to about 350 mM, or about 150 mM to about 350mM, or about 200 mM to about 350 mM, or about 250 mM to about 350 mM, orabout 300 mM to about 350 mM, or from about 0 mM to about 300 mM, orfrom about 10 mM to about 300 mM, or about 25 mM to about 300 mM, orabout 50 mM to about 300 mM, or about 75 mM to about 300 mM, or about100 mM to about 300 mM, or about 150 mM to about 300 mM, or about 200 mMto about 300 mM, or about 250 mM to about 300 mM, or from about 0 mM toabout 250 mM, or from about 10 mM to about 250 mM, or about 25 mM toabout 250 mM, or about 50 mM to about 250 mM, or about 75 mM to about250 mM, or about 100 mM to about 250 mM, or about 150 mM to about 250mM, or about 200 mM to about 250 mM, or from about 0 mM to about 200 mM,or from about 10 mM to about 200 mM, or about 25 mM to about 200 mM, orabout 50 mM to about 200 mM, or about 75 mM to about 200 mM, or about100 mM to about 200 mM, or about 150 mM to about 200 mM, or from about 0mM to about 150 mM, or from about 10 mM to about 150 mM, or about 25 mMto about 150 mM, or about 50 mM to about 150 mM, or about 75 mM to about150 mM, or about 100 mM to about 150 mM, or from about 0 mM to about 100mM, or from about 10 mM to about 100 mM, or about 25 mM to about 100 mM,or about 50 mM to about 100 mM, or about 75 mM to about 100 mM, or fromabout 0 mM to about 75 mM, or from about 10 mM to about 75 mM, or about25 mM to about 75 mM, or about 50 mM to about 75 mM, or from about 0 mMto about 50 mM, or from about 10 mM to about 50 mM, or about 25 mM toabout 50 mM, or from about 0 mM to about 25 mM, or from about 10 mM toabout 25 mM, or from about 25 mM to about 50 mM, or from about 25 mM toabout 100 mM, or from about 10 mM to about 100 mM, or from about 10 mMto about 75 mM, or from about 10 mM to about 50 mM, or from about 25 mMto about 375 mM. Preferably the concentration of Tris is from about 50mM to about 375 mM, and preferably 150 mM.

Sodium Dodecyl Sulfate or Other Anionic Surfactants

Sodium dodecyl sulfate (SDS) (C₁₂H₂₅SO₄Na) is an anionic surfactant iscommonly used in preparing proteins for electrophoresis in the SDS-PAGEtechnique. The molecule has a tail of 12 carbon atoms, attached to asulfate group, giving the molecule the amphiphilic properties requiredof a detergent.

SDS may be optionally added to the electrolyte solution of the presentinvention. The superior results obtained with the electrolyte solutionsaccording to the present invention are obtained independently of thepresence of SDS. The ranges of concentration over which SDS may be usedfor the preparation of electrolyte solutions according to the presentinvention, for all the applications discussed herein are from about 0.5%or less or from about 0.4% or less or from about 0.3% or less or fromabout 0.2% or less, or from about 0.1% or less, or from about 0.1% toabout 0.5%, or from about 0.2% to about 0.5%, or from about 0.3% toabout 0.5%, or from about 0.4% to about 0.5%, or from about 0.1% toabout 0.4%, or from about 0.2% to about 0.4%, or from about 0.3% toabout 0.4%, or from about 0.1% to about 0.3%, or from about 0.2% toabout 0.3%, or from about 0.1% to about 0.2%. Preferably, from about0.1% or less, and most preferably, at 0.1%.

Other anionic surfactants may be included in the electrolyte solution ofthe present invention, such as, in a non-limiting manner facultative,one anionic surfactant to provide to the buffer denaturing propertiesfor protein analysis: SDS, sodium dodecyl sulphate, lithium dodecylsulphate (LDS), sodium lauryl sulfate (SLS), sodium laurilsulfate orsodium, NaDS. The ranges of concentration over which they may be usedfor the preparation of electrolyte solutions according to the presentinvention, for all the applications discussed herein are from 1.0% orless. Preferably, from about 0.1% or less, and most preferably, at about0.1%.

Chelating Agents

Ethylenediaminetetraacetate (EDTA) has a role as a chelating agent, i.e.its ability to “sequester” metal ions such as Ca²⁺ and Fe³⁺. After beingbound by EDTA, metal ions remain in solution but exhibit diminishedreactivity. Other chelating agents may include ethylene glycoltetraacetic acid (EGTA), trisodium nitrilotriacetate, hydroxyethylethylenediamine trisodium acetate (trisodium HEDTA), diethylene triaminopentasodium acetate or uramil disodium acetate.

EDTA may be optionally added to the electrolyte solution of the presentinvention. The ranges of concentration over which EDTA may be used forthe preparation of electrolyte solutions according to the presentinvention, for all the applications discussed herein are from about 0.5%or less, or from about 0.4% or less, or from about 0.3% or less, or fromabout 0.2% or less, or from about 0.1% or less, or from about 0.05% orless, or from about 0.03% to about 0.5%, or from about 0.05% to about0.5% or from about 0.1% to about 0.5%, or from about 0.2% to about 0.5%,or from about 0.3% to about 0.5%, or from about 0.4% to about 0.5%, orfrom about 0.03% to about 0.4%, or from about 0.05% to about 0.4% orfrom about 0.1% to about 0.4%, or from about 0.2% to about 0.4%, or fromabout 0.3% to about 0.4%, or from about 0.03% to about 0.3%, or fromabout 0.05% to about 0.3% or from about 0.1% to about 0.3%, or fromabout 0.2% to about 0.3%, or from about 0.03% to about 0.5%, or fromabout 0.05% to about 0.2% or from about 0.1% to about 0.2%, or fromabout 0.03% to about 0.1%, or from about 0.05% to about 0.1%, or fromabout 0.03% to about 0.05%. Preferably, from about 0.05% or less, andmost preferably, at 0.03%.

Use of the Electrolyte Solutions

In use the electrolyte solutions of the present invention is compatiblewith a very wide range of other buffer systems. The electrolyte solutionof the present invention may be used to run electrophoresis of gels ofany type, prepared with the same or with different buffer systems thanthat of the present invention, even including gels using differentchemistries, such as MOPS as a buffer (such as those described in USPatent publication No. 20060118418), under the appropriate conditions.

According to the present invention, electrophoresis includes theseparation of samples of DNA or protein or any other type of moleculethat may be separated accordingly, as well as their transfer ontomembranes or other suitable solid support such has nitrocellulose,nylon, PVDF or other types of membranes that are commonly used forapplications such as Western transfer and blotting.

The electrolyte solution according to the present invention may be usedin electrophoresis chambers and/or systems of commercial make. Forexamples, Invitrogen™ Surelock™, BioRad™ Mini-Protean®, Protean® 2,Protean® μl, Protean® Tetra. Other equivalent systems also functions aswell.

The electrolyte solution of the present invention may be used as thebuffer system in most of gels used in molecular biology andbiochemistry, as described in classical references such as: Uriel 1966,Bull. Soc. Chem. Biol. 48:969; Peacock & Dingman 1967, Biochem 6(6),1818-1827; Peacock & Dingman 1968, Biochem 7(2), 668-674; Gaal,Electrophoresis in the separation of biological macromolecules, p 422,Wiley, 1980. The electrolyte solutions of the present invention may beincluded in acrylamide gels (polyacrylamide gels), under native (withoutSDS) or denaturing conditions (with SDS) that are typically preparedwith acrylamide concentrations from about 4% to about 25%. Theelectrolyte solutions of the present invention may be included inagarose gels that are typically prepared with agarose concentrationsfrom about 0.5% to about 3%.

EPPS (or HEPPS), TAPS, TES, BES or Glygly can be added to the Laemmlibuffer Tris-Glycine-SDS at a working concentration of 25 mM to 150 mMand preferably at 50 mM and extend the useful electrophoresis life ofelectrophoresis gels.

ALTERNATIVE EMBODIMENTS Example 1 Gel Electrophoresis of Novex® PrecastGels

An electrolyte solution according to the present invention is preparedto be used with polyacrylamide gels made using classic recipes such asthe Laemmli buffer system (Tris-Glycine-SDS). The exemplary electrolytesolution:

Zwitterion: EPPS: 6 g (47.5 mM), Tris (Base): 9 g (148.6 mM), SDS: 0.5 g(0.1%). The powders are dissolved in distilled water up to a volume of500 mL.

500 ml of running buffer were prepared with a zwitterion according tothe present invention as well as with a classic running buffer accordingto the Laemmli buffer system (Tris-Glycine-SDS). Age matched precast gelfrom NOVEX® (made with Tris pH 8.3 as buffering solution and glycine,according to the Leammli buffer system) that are pass their expiry date(as shown in FIG. 1) are run against each buffer. Protein molecularweight markers are separated on the electrophoresis gels. Theelectrophoresis is performed at 125V as recommended by the manufacturerof the gel. The duration of the electrophoretic run is recorded, and thequality gel migration is scored. After the electrophoretic run, themigration speed for the migration front to reach the bottom of the gel,and the resolution of a prestained molecular weight marker containing 10bands weighing between 15 KDa to 175 KDa is measured. The resolution ofeach of these bands of the gel run in the electrolyte solution accordingto the present invention is compared to the standard run in the Laemmlirunning buffer (Tris-Glycine-SDS, TGS). The results show that gels passtheir expiry date for over 3 years ran more than acceptably in thesolution of the present invention, while the older gels ran in theclassic TGS solution disintegrated during migration in the case of theoldest gel, or displayed clear migration artifacts (see the gel imagesin FIG. 1, last column in the second and fourth lines).

Example 2 Gel Electrophoresis of IDGel™ Precast Gels

Aged precast gels (IDGel™) made on Jun. 4, 2010 are kept at 4° C. untiluse on Jun. 10, 2011. The electrolyte solution according to the presentinvention is the same as in Example 1 (EPPS buffer), and it is comparedto the classic TGS Laemmli buffer (TGS buffer). The electrophoresis isperformed at 180V for 30 minutes (EPPS buffer) or for 40 minutes (TGSbuffer). After the electrophoretic run, the migration speed for themigration front to reach the bottom of the gel, and the resolution of aprestained molecular weight marker containing 10 bands weighing between15 KDa to 175 KDa is measured. The results show that the IDGel™separated in the EPPS buffer according to the present invention migratedwell and displayed sharp and well separated bands. The IDGel™ migratedin the control TGS buffer displayed distinctly fuzzier bands (See no. 20in FIG. 2). The gel matrix is also distorted during electrophoresis asthe gel expands and hangs out of the gel cassette (See no. 30 in FIG.2). Furthermore, bubbles are apparent between the glass plates of thecassette and the gel due to over-heating (See no. 10 in FIG. 2). Themeasured temperature in the TGS buffer is 65° C.

Example 3 Gel Electrophoresis of PAGEr™ Gold Precast Gels

Precast gels (PAGEr™ Gold from Lonza), having an 8-16% acrylamidegradient, 4 months post expiration date, are kept at 4° C. until use.The electrolyte solution according to the present invention is the sameas in Example 1 (EPPS buffer), and it is compared to the classic TGSLaemmli buffer (TGS buffer). The electrophoresis is performed at 300Vfor 21 minutes (EPPS buffer) or at 200V for 74 minutes (TGS buffer). Nowreferring to FIGS. 3A and B. After the electrophoretic run, themigration speed for the migration front to reach the bottom of the gel,and the resolution of prestained molecular weight markers and otherprotein samples is compared. The results show that overall, the PAGEr™Gold gel separated in the EPPS buffer according to the present invention(FIG. 3A) migrated well and displayed sharp and well separated bands.The PAGEr™ Gold gel migrated in the control TGS buffer displayeddistinctly fuzzier bands (See FIG. 3B). Since the voltage applied toeach gel is different, the PAGEr™ Gold gel separated in the control TGSbuffer is expected to complete its migration over a longer lapse oftime. However, the PAGEr™ Gold gel separated in the EPPS bufferaccording to the present invention completed its migration more rapidlythan would be expected had identical voltages been applied, with betterresolution than the PAGEr™ Gold gel separated in the control TGS buffer.

Example 4 Gel Electrophoresis of an EPPS Precast Gel

Now referring to FIG. 4. A precast gels (12% acrylamide), containing anEPPS based buffer, having been stored for 13 months at 4° C. until use,is separated in the electrolyte solution according to the presentinvention is the same as in Example 1 (EPPS buffer). The electrophoresisis performed at 225V for 19 minutes. Now referring to FIG. 4, after theelectrophoretic run, the migration speed for the migration front toreach the bottom of the gel, and the resolution of prestained molecularweight markers and other protein samples is evaluated. The results showthat overall gel separated in the EPPS buffer according to the presentinvention migrated well and displayed sharp and well separated bandsdespite its age.

The embodiments and examples presented herein are illustrative of thegeneral nature of the subject matter claimed and are not limiting. Itwill be understood by those skilled in the art how these embodiments canbe readily modified and/or adapted for various applications and invarious ways without departing from the spirit and scope of the subjectmatter disclosed claimed. The claims hereof are to be understood toinclude without limitation all alternative embodiments and equivalentsof the subject matter hereof. Phrases, words and terms employed hereinare illustrative and are not limiting. Where permissible by law, allreferences cited herein are incorporated by reference in their entirety.It will be appreciated that any aspects of the different embodimentsdisclosed herein may be combined in a range of possible alternativeembodiments, and alternative combinations of features, all of whichvaried combinations of features are to be understood to form a part ofthe subject matter claimed.

The invention claimed is:
 1. An electrolyte solution for extending auseful electrophoresis life of an electrophoresis gel comprising: atleast one zwitterion chosen from 2-amino-2methyl-1,3-propanediol (AMPD),N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid(AMPSO), N-Glycylglycine (Gly-Gly),4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or HEPPS),3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO),2-(cyclohexylamino)ethanesulfonic acid (CHES),N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),(2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino] ethanesulphonic acid)(TES), N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS),3-N-Morpholino propanesulfonic acid (MOPS), andPiperazine-N,N′-bis(2-hydroxypropanesulfonic acid) (POPSO); water;Tris(hydroxymethyl)aminomethane (TRIS), wherein the concentration ofTris(hydroxymethyl)aminomethane (TRIS) is from about 10 mM to about 500mM; and a chelating agent selected from the group consisting of ethyleneglycol tetraacetic acid (EGTA), trisodium nitrilotriacetate,hydroxyethyl ethylenediamine trisodium acetate (trisodium HEDTA),diethylene triamino pentasodium acetate or uramil disodium acetate,wherein the concentration of said chelating agent is from about 0.03% toabout 0.5% (wt/vol); wherein the pH of the electrolyte solution is fromabout 8.0 to about 11.0.
 2. The electrolyte solution of claim 1, whereinthe zwitterion is 4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid(EPPS or HEPPS).
 3. The electrolyte solution of claim 1, wherein thezwitterion is chosen from N-Glycylglycine (Gly-Gly) and3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO).
 4. Theelectrolyte solution of claim 1, further comprising sodium dodecylsulphate (SDS).
 5. The electrolyte solution of claim 1, wherein theconcentration of the zwitterion is from about 1 mM to about 500 mM. 6.The electrolyte solution of claim 5, wherein the concentration of thezwitterion is from about 10 mM to about 500 mM.
 7. The electrolytesolution of claim 5, wherein the concentration of the zwitterion is fromabout 25 mM to about 75 mM.
 8. The electrolyte solution of claim 5,wherein the concentration of the zwitterion is from about 50 mM to about100 mM.
 9. The electrolyte solution of claim 8, wherein theconcentration of the zwitterion is 50 mM.
 10. The electrolyte solutionof claim 8, wherein the concentration of the zwitterion is 100 mM. 11.The electrolyte solution of claim 4, wherein the concentration of sodiumdodecyl sulphate (SDS) is 0.5% (wt/vol) or less.
 12. The electrolytesolution of claim 11, wherein the concentration of sodium dodecylsulphate (SDS) is 0.1% (wt/vol) or less.
 13. The electrolyte solutionclaim 11, wherein the concentration of sodium dodecyl sulphate (SDS) is0.1% (wt/vol).
 14. The electrolyte solution claim 1, wherein theconcentration of said chelating agent is from about 0.05% to about 0.4%(wt/vol).
 15. A method of extending a useful electrophoresis life of anelectrophoresis gel during an electrophoretic separation of at least onesample comprising the step of: applying a voltage to an electrolytesolution according to claim 1, in contact with an electrophoresis gelcontaining the at least one sample therein.
 16. A method of extending auseful electrophoresis life of an electrophoresis gel during anelectrophoretic separation of at least one sample comprising the stepof: adding to an electrophoresis buffer containing an electrolytesolution according to claim 1 at least one different zwitterion chosenfrom 2-amino-2methyl-1,3-propanediol (AMPD),N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid(AMPSO), N-Glycylglycine (Gly-Gly),4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or HEPPS),3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO),2-(cyclohexylamino)ethanesulfonic acid (CHES),N,N-bis[2-hydroxyethyl]-2-aminoethanesulphonic acid (BES),(2-[2-hydroxy-1,1-bis(hydroxymethyl)ethylamino] ethanesulphonic acid)(TES), N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS)and 3-N-Morpholino propanesulfonic acid (MOPS).
 17. The method accordingto claim 16, wherein said zwitterion is4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS or HEPPS). 18.The method according to claim 16, wherein the zwitterion is chosen fromN-Glycylglycine (Gly-Gly) and3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO).
 19. Themethod according to claim 16, further comprising the step of applying avoltage to said electrophoresis buffer in contact with anelectrophoresis gel containing the at least one sample therein.